Photoreceptor development and survival requires the efficient transport of proteins from the inner segment to the outer segment. The central hypothesis of this proposal states that intraflagellar transport (IFT) proteins are essential for outer segment formation and protein transport through the connecting cilium and mutations in IFT genes cause retinal degeneration. The IFT proteins form a multisubunit complex that is transported along microtubules in the connecting cilium via the action of kinesin motors. We will use zebrafish as an experimental system because zebrafish photoreceptors are well-characterized, established techniques exist in zebrafish to manipulate gene expression and create genetic mosaic embryos, and we have zebrafish mutants for three IFT genes. These mutants exhibit mislocalized rhodopsin, disrupted photoreceptor outer segment formation, and photoreceptor degeneration. Our objective is to understand the function and regulation of the IFT particle in ciliary protein transport within the photoreceptor. Specifically, we propose to: 1) To determine the function and effects of mutations of three IFT proteins on zebrafish photoreceptor development by examining outer segment structure and protein localization using electron microscopy and light microscopy combined with immunohistochemistry. 2) To examine the regulation of IFT particle assembly and function by the Par-aPKC polarity complex. The IFT particle and Par-aPKC complex co-localize within the cilium and both interact with kinesin-ll. We will investigate the interactions between these complexes using immunoprecipitation and genetic mosaic analysis. 3) To measure IFT complex stability and movement in IFT57 mutants by analyzing the localization and movement of IFT-GFP fusion proteins assembled in the IFT particle using fluorescence microscopy and time-lapse confocal microscopy. 4) To determine the requirement for each IFT protein in photoreceptor morphogenesis by using morpholino oligonucleotides to generate Bardet-Biedl Syndrome phenotypes in zebrafish and assess photoreceptor development by light and fluorescence microscopy. As the mechanisms of protein transport required for photoreceptor development also function in the maintenance of adult photoreceptors, our studies can provide insights in the processes that lead to retinal pathologies. ? ? ?
Showing the most recent 10 out of 22 publications
